Cell observation device and cell observation method

ABSTRACT

A cell observation device is provided with a reflection interference measurement light source  106 , a quantitative phase measurement light source  105 , a reflection interference detection camera  110  which images light emitted from the reflection interference measurement light source  106  and reflected from cells  101 , to generate a reflection interference image, a quantitative phase detection camera  112  which images light emitted from the quantitative phase measurement light source  105  and transmitted by the cells  101 , to generate a quantitative phase image, an image alignment unit  201  which matches a spatial position of the reflection interference image with a spatial position of the quantitative phase image, to implement alignment between the two images, a first extraction unit  204  which extracts a first parameter from the reflection interference image in alignment with the quantitative phase image, and a second extraction unit  205  which extracts a second parameter from the quantitative phase image in alignment with the reflection interference image.

TECHNICAL FIELD

The present invention relates to a cell observation device and a cellobservation method.

BACKGROUND ART

Conventionally, a cell state was determined and evaluated by an invasivemethod, for example, by attaching a fluorescence label to cells.However, since such a method uses a toxic pigment for the cells or,fixes and treats the cells with a fixative solution, the cells cannot bereused after the treatment, and it is thus difficult to evaluate thecells in a natural state, to continue cultivation after evaluation, andto use the cells for transplantation. Therefore, it can be said that thecell state is preferably determined and evaluated by a non-invasivemethod. Patent Literature 1 and Non Patent Literatures 1 to 3 disclosedetermination and evaluation of cells by non-invasive methods.

CITATION LIST Patent Literature

-   Patent Literature 1: International Publication WO2005/029413

Non Patent Literatures

-   Non Patent Literature 1: Y. Usson, A. Guignandon, N. Laroche, M-H.    Lafage-Proust, L. Vico, “Quantitation of Cell-Matrix Adhesion Using    Confocal Image Analysis of Focal Contact Associated Proteins and    Interference Reflection Microscopy,” Cytometry, 28, 298-304, (1997)-   Non Patent Literature 2: C. K. Choi, C. H. Margraves, A. E.    English, K. D. Kihm, “Multicontrast microscopy technique to    dynamically fingerprint live-cell focal contacts during exposure and    replacement of cytotxic medium,” J. Biomedical Optics, 13 (5),    (2008)-   Non Patent Literature 3: I. Weber, R. Albrecht, “Image processing    for combined bright-field and reflection interference contrast video    microscopy,” Computer Methods and Programs in Biomedicine, 53,    113-118, (1997)

SUMMARY OF INVENTION Technical Problem

Patent Literature 1 discloses a method using a quantitative phasemicroscope, as one of methods for determining and evaluating the cellstate by a non-invasive method. This method allows non-invasivedetermination and evaluation on the cell state using a difference inoptical path length depending upon the presence/absence of cell.However, information obtained by this method is limited to onlyinformation based on the optical thickness, area, and volume of cell,and a change in refractive index in cell, which is not necessarily anamount of necessary information enough to appropriately determine andevaluate the cell state.

Non Patent Literatures 1 to 3 disclose methods using a reflectioninterference microscope, as another example of the non-invasive methods.These methods allow non-invasive determination and evaluation on thecell state by making use of a phenomenon in which a contrast of brightand dark patterns appears because of interference between reflectedlight from an interface between a substrate and a culture solution andreflected light from an interface between the culture solution and cellmembranes, based on adhesion states of individual cells to thesubstrate. However, the information obtained by these methods is limitedto only information based on the adhesion states of cells to thesubstrate, which is not necessarily an amount of necessary informationenough to appropriately determine and evaluate the cell state.

As described above, the methods using either the quantitative phasemicroscope or the reflection interference microscope singly can obtainonly the information based on the optical thickness, area, and volume ofcell, and the change in refractive index in cell, or only theinformation based on the adhesion states of cells to the substrate. Suchsingle information only cannot be an amount of necessary informationenough to appropriately determine and evaluate the cell state. We foundno literatures disclosing or suggesting combinational use of thequantitative phase microscope and the reflection interferencemicroscope, and the combinational use of the two microscopes mustrequire some devising because the two microscopes are separatemicroscopes with different functions.

The present invention has been accomplished in view of the abovecircumstances and it is an object of the present invention to provide acell observation device and a cell observation method enablingacquisition of a greater amount of information for appropriatedetermination and evaluation on the cell state.

Solution to Problem

In order to solve the above problem, a cell observation device of thepresent invention is one comprising: a reflection interferencemeasurement light source; a quantitative phase measurement light source;reflection interference imaging means which images light emitted fromthe reflection interference measurement light source and reflected froma cell, to generate a reflection interference image; quantitative phaseimaging means which images light emitted from the quantitative phasemeasurement light source and transmitted by the cell, to generate aquantitative phase image; image alignment means which matches a spatialposition of the reflection interference image with a spatial position ofthe quantitative phase image, thereby to implement alignment between thetwo images; first extraction means which extracts a first parameter fromthe reflection interference image in the alignment with the quantitativephase image by the image alignment means; and second extraction meanswhich extracts a second parameter from the quantitative phase image inthe alignment with the reflection interference image by the imagealignment means.

A cell observation method of the present invention is one comprising: areflection interference imaging step wherein reflection interferenceimaging means images light emitted from a reflection interferencemeasurement light source and reflected from a cell, to generate areflection interference image; a quantitative phase imaging step whereinquantitative phase imaging means images light emitted from aquantitative phase measurement light source and transmitted by the cell,to generate a quantitative phase image; an image alignment step whereinimage alignment means matches a spatial position of the reflectioninterference image with a spatial position of the quantitative phaseimage, thereby to implement alignment between the two images; a firstextraction step wherein first extraction means extracts a firstparameter from the reflection interference image in the alignment withthe quantitative phase image by the image alignment means; and a secondextraction step wherein second extraction means extracts a secondparameter from the quantitative phase image in the alignment with thereflection interference image by the image alignment means.

The cell observation device and the cell observation method of thepresent invention as described above, comprise the reflectioninterference measurement light source, the reflection interferenceimaging means, and the first extraction means (which will be referred tohereinafter as “reflection interference measurement unit”), whereby thefirst parameter is obtained based on the reflected light from the cell.Furthermore, the device and method comprise the quantitative phasemeasurement light source, the quantitative phase imaging means, and thesecond extraction means (which will be referred to hereinafter as“quantitative phase measurement unit”), whereby the second parameter isobtained based on the transmitted light from the cell. In this manner,the cell observation device and the cell observation method of thepresent invention can obtain both of the first parameter and the secondparameter by comprising both of the reflection interference measurementunit and the quantitative phase measurement unit, whereby a user obtainsa greater amount of information for appropriately determining andevaluating a state of the cell. Furthermore, the first parameter is aparameter extracted from the reflection interference image in thealignment with the quantitative phase image and the second parameter isa parameter extracted from the quantitative phase image in the alignmentwith the reflection interference image; therefore, it can be said thatthe two parameters have consistency as to a location where theparameters are extracted in the cell. Namely, the alignment between thetwo images by the image alignment means of the present invention enablesthe two parameters to be utilized as parameters for determining andevaluating the state at the same location of the cell.

In the present invention, the device may further comprise: contourextraction means which extracts a contour of the cell, based on thequantitative phase image; contour application means which applies thecontour extracted by the contour extraction means, to the reflectioninterference image to generate a reflection interference image aftercontour application; and third extraction means which extracts a thirdparameter from the reflection interference image after contourapplication.

In the present invention, the method may further comprise: a contourextraction step wherein contour extraction means extracts a contour ofthe cell, based on the quantitative phase image; a contour applicationstep wherein contour application means applies the contour extracted bythe contour extraction means, to the reflection interference image, togenerate a reflection interference image after contour application; anda third extraction step wherein third extraction means extracts a thirdparameter from the reflection interference image after contourapplication.

According to this invention, the third parameter is further obtained inaddition to the first parameter and the second parameter. Since thisthird parameter is a parameter obtained after matching of the cellcontour between the quantitative phase image and the reflectioninterference image, it is different in property from the first parameterand the second parameter; when further obtaining this third parameter,the user obtains a greater amount of information for appropriatelydetermining and evaluating the state of the cell.

There is no user's intervention in the process of extracting the contourof the cell from the quantitative phase image and applying the contourto the reflection interference image to generate the reflectioninterference image after contour application. On the other hand, theconventional method using the reflection interference microscope (whichwill be referred to hereinafter as “reflection interference method”)requires preliminary recognition of the contour of each individual cell,and therefore the reflection interference method is often used incombination with another method for contour recognition. Non PatentLiterature 1 above discloses the combination of the reflectioninterference method with the fluorescence method, but an operatordefines cell contours by handwriting because it was difficult toautomatically extract the cell contours. Non Patent Literature 2discloses the combination of the reflection interference method with thetransmission illumination method, but teaches nothing about automaticdetermination of cell contours without the aid of human hand.Furthermore, Non Patent Literature 3 discloses the combination of thereflection interference method with the bright-field method, andmentions automatization of extraction of cell contour, more or less;however, it describes, for example, that the user needs to manually setan optimum threshold for contour extraction while viewing a bright-fieldimage, thereby admitting the necessity for user's intervention to someextent and thus failing in automatically determining the cell contourswithout the aid of human hand. In contrast to it, in the presentinvention, the processing including the cell contour extraction from thequantitative phase image, the contour application to the reflectioninterference image, and the generation of the reflection interferenceimage after contour application is carried out by the contour extractionmeans and the contour application means, without the aid of human hand.When these processes are carried out without the aid of human hand, workefficiency is remarkably improved and in conjunction therewith anoperation time is significantly reduced.

In the present invention, the first parameter may be information basedon an adhesion state between a substrate on which the cell is laid, andthe cell.

According to this invention, the obtained information based on theadhesion state of the cell to the substrate includes such information asan adhesion area of the cell to the substrate, a ratio of the adhesionarea to an overall imaging range, and an adhesion condition (pattern ortwo-dimensional distribution) of the cell to the substrate, and the useris thus allowed to appropriately determine and evaluate the state of thecell, using these pieces of information.

In the present invention, the second parameter may be information basedon an optical thickness, area, and/or volume of the cell, or a change inrefractive index in the cell.

According to this invention, the obtained information includesinformation based on the optical thickness, area, and/or volume of thecell, or the change in refractive index in the cell, and the user isthus allowed to appropriately determine and evaluate the state of thecell, using these pieces of information.

In the present invention, the third parameter may be information basedon the adhesion state to the substrate, in the contour of the cell.

According to this invention, the obtained information based on theadhesion state to the substrate in the contour of the cell, i.e., in therange of a space occupied by the cell is information such as a ratio ofan adhesion area to an overall area of the cell, and the user is thusallowed to appropriately determine and evaluate the state of the cell,using such information.

In the present invention, the device may further comprise: referencestorage means which stores as reference data a parameter preliminarilyextracted for the cell of a known type or state; and analysis meanswhich determines a type or state of an unknown cell, based on thereference data.

According to this invention, the type or state of the cell as an unknownspecimen can be determined based on the reference data.

In the present invention, the device may further comprise analysis meanswhich selects a predetermined parameter from parameters extracted for anunknown cell and which determines a type or state of the unknown cell,using the predetermined parameter selected.

According to this invention, the analysis means can perform processing,without depending on the reference data. This configuration can berealized without the reference storage means and thus the deviceconfiguration becomes simpler. The analysis means may be configured toautomatically select a parameter indicative of a peculiar value as thepredetermined parameter, or may be configured to select thepredetermined parameter, based on an input from the user.

In the present invention, the device may further comprise: analysismeans which, when the first extraction means or the second extractionmeans extracts three or more parameters, performs a principal componentanalysis on the three or more parameters, thereby to determine a type orstate of an unknown cell.

According to this invention, the principal component analysis is carriedout on the large number of extracted parameters, whereby the largenumber of parameters can be made to suitably affect the celldetermination. The reason for it is that the principal componentanalysis is to perform the cell determination with two principalcomponents suitably reflecting all of the large number of parameters orsome of the three or more parameters, instead of selecting two out ofthe large number of extracted parameters.

In the present invention, low-coherent light may be used as thequantitative phase measurement light source.

According to this invention, the low-coherent light with a widewavelength band and with low coherency is used in the quantitative phasemeasurement. This reduces interference noise originating in an opticalsystem and thus allows stabler measurement.

In the present invention, low-coherent light may be used as thereflection interference measurement light source.

According to this invention, the low-coherent light with a widewavelength band and with low coherency is used in the reflectioninterference measurement. When the illumination light used is one with awide wavelength band, the distance of occurrence of interference can bedecreased and the reflection interference image can be taken as beinglimited to an adhesion face of the cell to the substrate.

In the present invention, the device may further comprise: an objectivelens which condenses the light emitted from the reflection interferencemeasurement light source and reflected from the cell; and a slit of aring shape at a position conjugate with an aperture stop on thereflection interference measurement light source side of the objectivelens.

According to this invention, the illumination light from the reflectioninterference measurement light source passes through the slit opening inthe ring shape, and the illumination light passes through the peripherywithout passing through the center of the objective lens, to illuminatethe cell; therefore, the cell is illuminated with only the angled lightwith high NA, which can reduce influence of reflected light from asolution above the cell. The use of the slit of the ring shape can alsoreduce background light due to reflection inside the objective lens.

In the present invention, the device may further comprise a vessel whichhouses the cell, and an antireflection coat may be laid on the side ofthe vessel opposite to an adhesion face of the cell.

According to this invention, the reflection interference image of thecell adhesion face can be obtained with high contrast even in the caseusing the objective lens of a dry type.

In the present invention, the device may comprise an observation windowat such a height as to contact a culture solution in which the cell isimmersed, in an upper part of the vessel.

According to this invention, the height of the culture solution is keptconstant with the use of the observation window, whereby thequantitative phase measurement can be carried out under a stablecondition.

In the present invention, the observation window may be provided with anantireflection coat on the side thereof opposite to a surface in contactwith the culture solution.

According to this invention, the reflection interference image of thecell adhesion face can be obtained with high contrast, while suppressinginfluence of reflected light from a top surface of the vessel.

In the present invention, the device may further comprise a mirrorlocated between the reflection interference measurement light source andthe reflection interference imaging means and between the quantitativephase measurement light source and the reflection interference imagingmeans and having variable ratios of reflection to transmission dependingupon wavelengths.

According to this invention, the device comprises the mirror having thevariable ratios of reflection to transmission depending uponwavelengths, whereby, for example, when the wavelengths of thequantitative phase measurement light source are different from those ofthe reflection interference measurement light source, the device can beconfigured to set such a ratio of reflection to transmittance as to makethe mirror function as a half mirror for the wavelengths of thereflection interference measurement light source and to increase thetransmittance according to the wavelengths of the quantitative phasemeasurement light source. This configuration allows the mirror tofunction as a half mirror in the reflection interference measurement andto reduce a loss of observation light in the quantitative phasemeasurement.

Advantageous Effect of Invention

The present invention can provide the cell observation device and thecell observation method capable of obtaining the greater amount ofinformation for appropriately determining and evaluating the cell state.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view showing an overall configuration of a cellobservation device 1.

FIG. 2 is a hardware configuration diagram of a processing unit 20.

FIG. 3 is a flowchart showing functions and operation of the processingunit 20.

FIG. 4 is a drawing showing a sample 30 used in image alignment.

FIG. 5 is a drawing showing an example of a contour extraction processand a contour application process.

FIG. 6 is a drawing showing an example of parameters obtained fromcells.

FIG. 7 is an example of two-component scatter diagrams created usingextracted parameters.

FIG. 8 is a drawing showing an example in which cell determination wasconducted using reference data.

FIG. 9 is a drawing for showing an effect by a third example.

FIG. 10 is a schematic diagram of a configuration of a cell observationdevice 1A according to a fourth example.

FIG. 11 is a drawing for showing an effect by the fourth example.

FIG. 12 is a drawing showing a vessel 102A in a fifth example.

FIG. 13 is a drawing for showing an effect by the fifth example.

FIG. 14 is a drawing for showing an effect by the fifth example.

FIG. 15 is a drawing for showing an effect by the fifth example.

FIG. 16 is a drawing showing a modification example of the fifthexample.

FIG. 17 is a drawing showing an image acquisition unit 10A in a sixthexample.

FIG. 18 is a drawing showing an image acquisition unit 10B in a seventhexample.

FIG. 19 is a drawing showing an image acquisition unit 10C in an eighthexample.

FIG. 20 is a drawing showing an image acquisition unit 10D in a ninthexample.

FIG. 21 is a drawing showing an image acquisition unit 10E in a tenthexample.

FIG. 22 is a drawing showing an image acquisition unit 10F in aneleventh example.

FIG. 23 is a drawing showing an image acquisition unit 10G in a twelfthexample.

FIG. 24 is a drawing showing an image acquisition unit 10H in athirteenth example.

FIG. 25 is a drawing showing an image acquisition unit 10I in afourteenth example.

FIG. 26 is a drawing showing an example of a screen displayed for a userin a fifteenth example.

FIG. 27 is a drawing showing reflection interference images andquantitative phase images of respective cells used as reference, in asixteenth example.

FIG. 28 is a drawing showing actually measured values of seven extractedparameters, in the sixteenth example.

FIG. 29 is a drawing showing the result of calculation of the sum ofsquares of deviations from a population as an information amount ofparameters, in the sixteenth example.

FIG. 30 is a drawing showing the result of standardization for each ofparameters, in the sixteenth example.

FIG. 31 is a drawing showing the result of calculation of coefficientsfor a first principal component f, in the sixteenth example.

FIG. 32 is a drawing showing the result of calculation of coefficientsfor a second principal component g, in the sixteenth example.

FIG. 33 is a drawing showing the result of calculation of the firstprincipal component f and the second principal component g for each ofcells, in the sixteenth example.

FIG. 34 is a drawing showing the result of plotting of each of cells ona two-component scatter diagram, using values of the first principalcomponent f and the second principal component g, in the sixteenthexample.

DESCRIPTION OF EMBODIMENTS

The preferred embodiments of the cell observation device and the cellobservation method according to the present invention will be describedbelow in detail with reference to the accompanying drawings. The cellobservation device and the cell observation method will be firstgenerally outlined and then they will be described in more detail usingthe sections of first to sixteenth examples. In the description of thedrawings the same elements will be denoted by the same reference signs,without redundant description.

[Overall Configuration of Cell Observation Device 1]

First, an overall configuration of a cell observation device 1 accordingto an embodiment of the present invention will be described withreference to FIG. 1. FIG. 1 is a schematic view showing the overallconfiguration of the cell observation device 1. As shown in FIG. 1, thecell observation device 1 is composed of an image acquisition unit 10and a processing unit 20.

The image acquisition unit 10 is provided with a vessel 102 housingcells 101 as a sample, a culture space 103 maintained in a cultureenvironment for the cells 101, an objective lens 104, a quantitativephase measurement light source 105, an illumination stop unit 105A, areflection interference measurement light source 106, a half mirror 107,a dichroic mirror 108, a total reflection mirror 109, a reflectioninterference detection camera 110 (corresponding to “reflectioninterference imaging means” in the scope of claims), a diffractiveinterference optical system 111, and a quantitative phase detectioncamera 112 (corresponding to “quantitative phase imaging means” in thescope of claims). The half mirror 107 is an optical system forreflection interference incidence that is provided for guiding lightfrom the reflection interference measurement light source 106 to thecells 101, and the dichroic mirror 108 is an optical system forreflection interference measurement that is provided for guiding lightfrom the cells 101 to the reflection interference detection camera 110.The objective lens 104, the half mirror 107, and the dichroic mirror 108constitute a common optical system for guiding light from an identicalzone of the cells 101 to the reflection interference detection camera110 and to the quantitative phase detection camera 112. Namely, in thepresent embodiment the reflection interference incidence optical systemand the reflection interference measurement optical system areconstructed as the common optical system. Furthermore, the totalreflection mirror 109 and the diffractive interference optical system111 constitute a quantitative phase optical system for guiding light tothe quantitative phase detection camera 112. The reflection interferencedetection camera 110 images the light emitted from the reflectioninterference measurement light source 106 and reflected by the cells101, to generate a reflection interference image (which corresponds to“reflection interference imaging step” in the scope of claims). Thequantitative phase detection camera 112 images the light transmitted bythe cells 101, of the illumination light emitted from the quantitativephase measurement light source 105 and passed through the illuminationstop unit 105B such as a pinhole or an aperture, so as to turn intoillumination light close to a point light source, thereby to generate aquantitative phase image (which corresponds to “quantitative phaseimaging step” in the scope of claims). The quantitative phasemeasurement light source 105 to be used can be a light source withradiation sensitivity over a wide wavelength band such as a halogen lampor a xenon lamp. The light source to be used may be one such as an LED(light emitting diode), a semiconductor laser (laser diode), or an SLD(super luminescent diode). In the case of the laser or the SLD, theillumination stop unit 105A can be omitted because the light source hasa sufficiently small size.

The processing unit 20 is provided with a first processing unitconsisting of an image alignment unit 201 (corresponding to “imagealignment means” in the scope of claims), a first extraction unit 204(corresponding to “first extraction means” in the scope of claims), anda second extraction unit 205 (corresponding to “second extraction means”in the scope of claims), a second processing unit consisting of acontour extraction unit 202 (corresponding to “contour extraction means”in the scope of claims), a contour application unit 203 (correspondingto “contour application means” in the scope of claims), and a thirdextraction unit 206 (corresponding to “third extraction means” in thescope of claims), and a third processing unit consisting of an analysisunit 207 (corresponding to “analysis means” in the scope of claims) anda reference storage unit 208 (corresponding to “reference storage means”in the scope of claims). The first processing unit, the secondprocessing unit, and the third processing unit may be constructed asrespective separate arithmetic devices or as a single arithmetic device.

The image alignment unit 201 is a unit that matches the spatial positionof the reflection interference image with that of the quantitative phaseimage, thereby to implement alignment between the two images(corresponding to “image alignment step” in the scope of claims). Thecontour extraction unit 202 is a unit that extracts contours as zones ofthe cells 101, based on the quantitative phase image (corresponding to“contour extraction step” in the scope of claims). The contourapplication unit 203 is a unit that applies the contours extracted bythe contour extraction means, to the reflection interference image, togenerate a reflection interference image after contour application(corresponding to “contour application step” in the scope of claims).

The first extraction unit 204 is a unit that extracts a first parameterfrom the reflection interference image in alignment with thequantitative phase image by the image alignment means (corresponding to“first extraction step” in the scope of claims). The first parameter isinformation based on an adhesion state between the cells 101 and asubstrate on which the cells 101 are laid. The information based on theadhesion state of the cells 101 to the substrate can be, for example,information such as an adhesion area of the cells 101 to the substrate,a ratio of the adhesion area to an overall imaging range, or an adhesioncondition (pattern or two-dimensional distribution) of the cells to thesubstrate.

The second extraction unit 205 is a unit that extracts a secondparameter from the quantitative phase image in alignment with thereflection interference image by the image alignment means(corresponding to “second extraction step” in the scope of claims). Thesecond parameter is information based on (or indicative of) an opticalthickness, an area, and/or a volume of cells 101, or a change inrefractive index in the cells 101.

The third extraction unit 206 is a unit that extracts a third parameterfrom the reflection interference image after contour application(corresponding to “third extraction step” in the scope of claims). Thethird parameter is information based on an adhesion state to thesubstrate, in the contours of the cells 101. The information based onthe adhesion state to the substrate in the range of the space occupiedby the cells 101 can be, for example, information such as a ratio of theadhesion area to the overall area of the cells 101.

The reference storage unit 208 is a unit that preliminarily stores asreference data, parameters (the aforementioned first, second, and thirdparameters) extracted for cells of known types or states. The analysisunit 207 is a unit that determines a type or a state of a cell as anunknown specimen, based on the first, second, and third parametersextracted by the first extraction unit 204, the second extraction unit205, and the third extraction unit 206. Specifically, the analysis unitselects a predetermined parameter from the first, second, and thirdparameters and determines a type or a state of a cell as an unknownspecimen, using the predetermined parameter thus selected. Thepredetermined parameter may be a parameter indicative of a peculiarvalue automatically selected by the analysis unit 207 (or anotherprocessing means not shown), or a parameter selected by a user. Theanalysis unit 207 determines the type or the state of the cell as theunknown specimen, using the predetermined parameter thus selected. Theanalysis unit 207 may be configured to select the predeterminedparameter, based on the reference data stored in the reference storageunit 208, and to determine the type or state of the cell as the unknownspecimen, using it. Furthermore, when each of the first extraction unit204, the second extraction unit 205, and the third extraction unit 206,or a combination thereof extracts three or more parameters, the analysisunit 207 may perform a principal component analysis on the three or moreparameters to determine the type or state of the unknown cell. In thedescription hereinafter, a mode in which the analysis unit 207 selectsthe predetermined parameter based on the reference data and determinesthe type or state of the cell as the unknown specimen will be describedin the section of [First Example]. On the other hand, a mode wherein theanalysis unit 207 selects the predetermined parameter in accordance witha peculiar value or a user command, without basis on the reference data,and determines the type or state of the cell as the unknown specimenwill be described in the section of [Fifteenth Example]. Furthermore, amode wherein the analysis unit 207 determines the type or state of theunknown cell by the principal component analysis will be described inthe section of [Sixteenth Example].

FIG. 2 is a hardware configuration diagram of the processing unit 20having the functional constituent elements as described above. As shownin FIG. 2, the processing unit 20 is constructed, physically, as anordinary computer system including a CPU 21, main memories such as ROM22 and RAM 23, input devices 24 such as a keyboard and a mouse, anoutput device 25 such as a display, a communication module 26 such as anetwork card for transmission and reception of data to and from theimage acquisition unit 10, and an auxiliary memory 27 such as a harddisk. Each of the functions of the processing unit 20 is substantializedin such a manner that predetermined computer software is retrieved ontothe hardware such as the CPU 21, ROM 22, and RAM 23 to make the inputdevices 24, output device 25, and communication module 26 operate undercontrol of the CPU 21 and data is read out and written into the mainmemories 22, 23 and the auxiliary memory 27.

First Example

The first example of the present invention will be described below indetail referring again to FIG. 1. FIG. 1 is the schematic configurationdiagram of the cell observation device 1 according to the first example.

(Description of Image Acquisition Unit 10)

As shown in FIG. 1, the vessel 102 with the cells 101 as a measurementtarget therein is stationarily placed in the culture space 103maintained in the culture environment for cells 101. The cultureenvironment is an environment at controlled temperature, humidity,carbon dioxide concentration, etc. suitable for development or statemaintenance of the cells 101.

The quantitative phase measurement will be described. The illuminationlight emitted from the quantitative phase measurement light source 105disposed above the vessel 102 with the cells 101 therein, travelsthrough the vessel 102 with the cells 101 therein, to be condensed bythe objective lens 104. Then the light travels via the half mirror 107,then passes through the dichroic mirror 108 for separation of thequantitative phase image and the reflection interference image bywavelengths, and further travels via the total reflection mirror 109 toform an interference image between object light and reference light inthe diffractive interference optical system 111 for phase measurement,and the interference fringe image is taken by the quantitative phasedetection camera 112.

The reflection interference measurement will be described. Theillumination light emitted from the reflection interference measurementlight source 106 is reflected by the half mirror 107, and then travelsthrough the objective lens 104 to enter the vessel 102 with the cells101 as a measurement target therein, from the bottom side thereof.Without having to be limited to the half mirror 107, a beam splitterwith a reduced reflection ratio, e.g., 5:95 (reflection:transmission) or20:80 (reflection:transmission), may also be used if the intensity ofthe illumination light is sufficiently high. It is also possible to usea dichroic mirror with the reflectance and transmittance differingdepending upon wavelengths. Reflected light from adhesion faces of thecells 101 on the bottom of the vessel 102 causes interference accordingto adhesion distances of the cells 101, and resultant reflectioninterference light is condensed again by the objective lens 104 andtravels via the half mirror 107; only the reflection interference imageis reflected by the dichroic mirror 108 for separation of thequantitative phase image and the reflection interference image bywavelengths, and picked up by the reflection interference detectioncamera 110. When a beam splitter such as a half mirror is used insteadof the dichroic mirror 108, a filter for selection of the wavelengths tobe used in reflection interference detection is to be placed between thebeam splitter and the reflection interference detection camera 110. Thelight reflected from the adhesion faces of the cells 101 on the bottomof the vessel 102 has different amplitudes of interfering lightdepending upon the adhesion distances of the cells 101 and is imaged asa contrast of bright and dark patterns. Since the quantitative phaseimage and the reflection interference image are acquired through thecommon objective lens 104, the imaging range of the cells 101 isapproximately the same in the quantitative phase measurement and thereflection interference measurement.

The camera 112 to acquire the quantitative phase image and the camera110 to acquire the reflection interference image do not have to belimited to cameras of the same performance and same pixel resolution.Since quantities and wavelengths of the light beams incident into therespective cameras are different from each other, they may be cameras ofperformances and spatial resolutions suitable for the respectiveoperations. For example, the quantitative phase detection camera 112 maybe a sensitivity-priority camera with high sensitivity to 830 nm andwith a large pixel size, and the reflection interference detectioncamera 110 may be a spatial-resolution-priority camera with highsensitivity to the visible range and with a small pixel size. Thisconfiguration requires a process of matching spatial coordinates of thetwo cameras, and this process will be described later.

In order to allow discrimination between the quantitative phasemeasurement and the reflection interference measurement by wavelengths,beams in different wavelength bands may be used for the respectiveillumination wavelengths of the reflection interference measurementlight source 106 and the quantitative phase measurement light source105. The dichroic mirror 108 can separate and select the quantitativephase image and the reflection interference image by the specificwavelengths. Accordingly, it becomes feasible to obtain the images atthe same time without crosstalk by the quantitative phase measurementand the reflection interference measurement.

For observing as many cells 101 as possible, the device may be providedwith a mechanism for moving the observation position. In order tominimize influence on the cells 101 and suppress vibration of the liquidlevel in the quantitative phase measurement, it is desirable to adopt amethod of changing the observation position by moving the main body ofthe image acquisition unit 10 as an integrated body of the illuminationoptical system and the observation optical system on the XY plane, whilekeeping the vessel with the cells 101 therein stationary. At the sametime, it is desirable to record plane coordinates in the XY space underobservation on the images.

(Description of Processing Unit 20)

The functions and operation of the processing unit 20 will be describedfurther referring to the flowchart of FIG. 3.

First, the quantitative phase detection camera 112 acquires theinterference fringe image between reference light and, object lighthaving passed through the cells 101 (step S101, corresponding to“quantitative phase imaging step” in the scope of claims). Aquantitative phase image is formed from the interference fringe image bya well-known arithmetic method. For obtaining the quantitative phaseimage, an offset correction of the background region without the cells101 and a shading correction in the field of the background region arecarried out to make the background part spatially uniform and correctthe phase value of the background part to 0, thereby obtaining atwo-dimensional map of phases (optical path lengths) of the cells 101.

On the other hand, in parallel with the step S101, the reflectioninterference detection camera 110 acquires the reflection interferenceimage of adhesion faces of the cells 101 (step S102, corresponding to“reflection interference imaging step” in the scope of claims). Sinceamplitudes of interference light are different depending upon distancesof the cells 101 adhering to the bottom surface of the vessel 102, fromthe bottom surface of the vessel 102, the reflection interference imageis taken as a contrast of bright and dark patterns. Correction is madefor shading of reflected light in the field of the reflectioninterference image. At the same time an offset correction for thebackground part is carried out in each time unit, in order to preventvalues of the background without the cells 101 from varying with time.Through these image arithmetic corrections, we can obtain thequantitative phase image and the reflection interference image withlittle spatial and temporal variations.

The next step is to perform a correction for spatial positions of thequantitative phase image and the reflection interference image (stepS103, corresponding to “image alignment step” in the scope of claims).FIG. 4 shows a sample 30 used in image alignment. The sample 30 foralignment to be used herein can be a micro scale in which a grid 31 isscribed on a glass substrate, or a dot pattern. When the micro scale orthe dot pattern is provided with at least one mark to identify the sameposition on images taken by the two cameras, the operation becomessimpler. In the example of FIG. 4, there are two marks, a triangle and adot.

A procedure for shifting the image of the reflection-interference-sidecamera 110 so as to be aligned in position with the quantitative phaseimage will be described below in detail. First, the micro scale or thedot pattern is placed instead of the sample of cells 101 and is taken byeach of the taking cameras for the reflection interference and thequantitative phase. Next, a reflection interference image and aquantitative phase image obtained by the photography of the micro scaleor the dot pattern are superimposed on each other in respectivedifferent false colors (e.g., green for the reflection interferenceimage and red for the quantitative phase image). In some cases theimages become easily viewable with inversion of luminance thereof. Next,in order to match the grid image or dot image of the reflectioninterference image with the grid image or dot image of the quantitativephase image, while viewing the superimposed images, the reflectioninterference image is finely adjusted in (1) enlargement/reductionratio, (2) horizontal movement amount (number of pixels), (3) verticalmovement amount (number of pixels), (4) angle of rotation, (5) directionof rotation, (6) mirror reversal, etc. to determine adjustment amountsthereof. The adjustment amounts can be determined to achieve suchposition that the grid images or dot images displayed in superpositionoverlap each other with no edges projecting out, for example, suchposition that the grid images or dot images overlap each other to turngreen and red into yellow.

If the two camera images taken have spatial distortions and patterns ofthe spatial distortions of the two images are largely different, it issometimes difficult to equally match the positions of all the pixels bythe aforementioned data (1) to (6) only. In such cases movement amountsare different for all the pixels in the field and it is thus necessaryto determine the movement amounts for the respective pixels.Specifically, shift amounts of respective points are determined so as tomatch intersections of the grid images or center coordinates ofrespective dots of the dot images with each other between the twocameras. Shift amounts for spatial coordinates except for thecoordinates where the grid or dots exist are determined byinterpolation. A table is created by storing the shift amounts in pixelunit for spatially all pixels (all coordinates) in this manner, and isdefined as alignment data. The alignment data thus obtained is stored asa file. When new measurement is performed to acquire images, alignmentbetween the images is carried out using the alignment data stored in thefile, and position-corrected images are output. When the image dataacquired through execution of new measurement is stored in a file, thealignment data is preferably stored together with the image data in thefile. This allows the device to quote the alignment data, which was thebest in new acquisition of the images, in calling of the image file andto correct the called images.

The alignment as described above does not have to be performed everymeasurement of quantitative phase and reflection interference, but maybe performed, for example, at the frequency of once per month, withconsideration to positional deviation due to influence of the useenvironment and vibration, as long as the same optical system is used.If the observation-side optical system is equipped with a component toselect a filter for observation (e.g., an electric filter wheel), thealignment is preferably performed for each filter to be used. The reasonfor it is that positional deviation amounts and directions of positionaldeviation of images differ depending upon inclination or parallelism ofthe filter to be used.

Although description is omitted to avoid redundancy, the above procedurecan also be suitably applied to the case opposite to the above, i.e.,the case where the image of the quantitative-phase-side camera 112 isshifted in position so as to match the reflection interference image.The above procedure can be carried out without the aid of human hand byautomated image processing.

Referring back to FIG. 3, after the alignment in the step S103, aprocess of extracting contour regions of the cells 101 (which will alsobe referred to hereinafter as “segmentation”) is carried out (steps S104and S105, corresponding to “contour extraction step” and “contourapplication means” in the scope of claims).

First, as shown in FIG. 5, regions as contours of individual cells 101are detected from the quantitative phase image out of the quantitativephase image and the reflection interference image after taken at thesame time and aligned with each other, by image processing (step S104,image A in FIG. 5, corresponding to “contour extraction step” in thescope of claims). Namely, in the quantitative phase image, the opticalpath lengths of light passing through the cells 101 become longer thanthose of light passing through the solution as the background withoutthe cells 101 therein, because the refractive index of the cells 101 islarger than that of the solution. For this reason, phase values ofpixels in the regions where the cells 101 exist become larger than thosein the background. Therefore, when an appropriate threshold or spatialfiltering process is applied, the cells 101 can be separated from thebackground automatically without the aid of human hand. Then thecontours corresponding to the respective cells 101 can be determined andregions of pixel coordinates corresponding to the regions occupied bythe respective cells 101 can be determined.

Next, the pixel coordinates of the contour regions of the individualcells 101 obtained in step S104 are adapted to the reflectioninterference image aligned in spatial coordinates, i.e., thesegmentation regions obtained in step S104 are copied onto thereflection interference image, whereby the contour regions of theindividual cells 101 determined on the quantitative phase image areapplied to the reflection interference image (step S105, image B in FIG.5, corresponding to “contour application step” in the scope of claims).By this step, as shown in FIG. 5, the same contour regions can bedetermined for the two images A, B of the quantitative phase image andthe reflection interference image.

After the segmentation in steps S104 and S105, a process of extractingparameters is then carried out (step S106, corresponding to “firstextraction step,” “second extraction step,” and “third extraction step”in the scope of claims).

Namely, in the regions of the respective cells 101 obtained in stepS104, the second extraction unit 205 acquires from the quantitativephase image, (1) an average value of optical thicknesses (optical pathlengths) of the cells 101, (2) a standard deviation of opticalthicknesses, (3) an area of the cells 101, and (4) an optical volume ofthe cells 101 (a total value of optical thicknesses) (step S106,corresponding to “second extraction step” in the scope of claims).Furthermore, from the reflection interference image aligned in step S103and the reflection interference image with the copy of the segmentationregions obtained in step S105, the first extraction unit 204 and thethird extraction unit 206 each obtain (5) an average luminance of brightand dark regions, (6) an area of dark regions indicating strongadhesion, (7) an area of bright regions indicating weak adhesion, (8)ratios of dark regions to the overall imaging range and to the area ofcells 101, and (9) ratios of bright regions to the overall imaging rangeand to the area of cells 101 (step S106, corresponding to “firstextraction step” and “third extraction step” in the scope of claims).

A quantitative analysis to analyze a texture obtained from thetwo-dimensional intensity distribution pattern of bright and darkregions of the reflection interference image is carried out as a methodto obtain a two-dimensional distribution pattern of adhesion portions.The texture analysis is performed by applying the co-occurrence matrixmethod to acquire features of the texture obtained from a co-occurrencematrix with elements of probabilities P(i,j) (i,j=0, 1, 2, 3, . . . n−1)that a gray level of a pixel distant by the distance of d pixels in theθ-direction from a pixel with a gray level i in the image is j, the graylevel histogram method to acquire features of the texture from a graylevel histogram P(i) in the image, and so on. This analysis process maybe carried out by the analysis unit 207.

Examples of feature parameters of the texture obtained by theco-occurrence matrix method include (10) energy, (11) entropy, (12)correlation, (13) local uniformity, (14) inertia, and so on. Amongthese, energy is an index indicating whether the probabilitydistribution of the co-occurrence matrix is concentrated at a specificvalue, and entropy an index indicating whether the probabilitydistribution of the co-occurrence matrix is distributed over values in awide range. On the other hand, examples of feature parameters of thetexture obtained from the gray level histogram method include (15)average, (16) variance, (17) skewness, (18) kurtosis, and so on. Amongthese, skewness is an index indicating how far the shape of the graylevel histogram deviates from a symmetric shape, and kurtosis an indexindicating how close the distribution of the gray level histogram isconcentrated around an average.

Since the parameters of (12) correlation by the co-occurrence matrixmethod and (15) average and (16) variance by the gray level histogrammethod among those are readily affected by brightness of luminosity uponacquisition of the image, evaluation had better be made by capturing acondition that they are limited to those acquired in the same exposureduration and at the same illumination luminance, for example. In thiscase, (19) “Π (variance)/average luminosity” can be used instead ofvariance, whereby it can be used as a parameter that is not affected bythe condition of brightness upon acquisition of the image. In theco-occurrence matrix method, we can obtain a plurality of values aboutthe direction θ and distance d, and a large number of parameters can beoptimized if a preliminary experiment is carried out in advance toinvestigate and determine differences of parameters among cells with anydirection and any distance. The result this time will be obtained byfixing the θ-direction=0° and the distance d=8 pixels. Numerical valuesof the parameters (1) to (19) about the individual cells 101 obtained inthis manner are stored as the measurement result.

FIG. 6 shows an example of the parameters obtained from the individualcells. A scatter diagram can be drawn using two components out of theseparameters. FIG. 7 is plots using characteristic parameters fordiscrimination as to two types of cells, myeloid stem cells anddifferentiation-induced osteoclasts. (A) in FIG. 7 is a two-componentscatter diagram as a plot of the respective types of cells in which thehorizontal axis represents the average luminosity (5) of bright and darkpatterns in reflection interference and the vertical axis the average(1) of optical thicknesses of cells in quantitative phase. On the otherhand, (B) in FIG. 7 is a plot in which the horizontal axis representsthe kurtosis (16) in the gray level histogram method of the textureanalysis in reflection interference and the vertical axis the inertia(13) in the co-occurrence matrix method of the texture analysis inreflection interference. In either of the two-component scatterdiagrams, stem cells and osteoclasts form their respective groups andthus the parameters are effective to discrimination of different typesof cells.

Referring back to FIG. 3, based on the measurement information(parameters) obtained from the individual cells 101 in step S106, it isdetermined to which type of cell group the cells as the unknown specimenare similar, or to which state of cell group the cells as the unknownspecimen are similar (step S107). For this determination, measurementinformation on known states of cell groups is preliminarily stored asreference data and the type or state of the cells as the specimen isdetermined by comparing the specimen cells with the reference data. Thecell observation device 1 is provided with the reference storage unit208 for storage of the reference data, as shown in FIG. 1. The referencestorage unit 208 can store a plurality of types of cells or a pluralityof types of states of cells, can also have a reference about a typicalcell group as a default value, and can also store additional dataobtained through execution of new measurement with a cell groupnecessary for an operator.

A procedure of acquiring the reference data includes first preparingmultiple cells from a known type of cell group or a known state of cellgroup in advance. These cells to be prepared should be cells with littlevariation among them and with quality as homogenous as possible. Next,the aforementioned processes of steps S101 to S106 are carried out forthese cells by collaboration of the image acquisition unit 10 and theprocessing unit 20 and the measurement result obtained is stored asreference data in the reference storage unit 208.

Let us consider a situation in which it is determined to which unknowncells as a specimen belong between two cell groups, as an example ofcell determination using the reference data. The following determinationprocess is carried out by the analysis unit 207. First, as to two typesof cell groups stored as reference, arbitrary two components areselected from the measurement result of (1) to (19) of the individualcells and two-component scatter diagrams are created with the twocomponents on the vertical axis and on the horizontal axis,respectively. Among the two-component scatter diagrams thus created, atwo-component scatter diagram to allow simple discrimination between thetwo cell groups by a simple partition line is used as determinationcriteria in discrimination of cells as a specimen. For example, in thecase of the reference data as an example shown in (A) of FIG. 8, ascatter diagram is obtained as means for discrimination between cells Aand cells B by plotting the kurtosis (16) in the gray level histogrammethod of the texture analysis in reflection interference on thehorizontal axis and plotting the inertia (13) in the co-occurrencematrix method of the texture analysis in reflection interference on thevertical axis. Use of this two-component scatter diagram allowsdiscrimination between cells A and cells B by a simple partition line L.The discrimination can be made with the sensitivity of 100% and thespecificity of 100%, as the performance of the discrimination of cells Awith this partition line L.

Next, measurement is carried out for unknown cells as a specimen andparameters of the measurement result of (1) to (19) described above areobtained for each of unknown cells. When discrimination is to determinewhether the cells as a specimen are cells A or cells B, thetwo-component scatter diagram of reference data for discriminationbetween cells A and cells B is called and reference is made thereto. Asshown in (B) and (C) of FIG. 8, the specimen cells are plotted on thetwo-component scatter diagram of reference data and the regionscorresponding to cells A and cells B are clearly shown using thepartition line L. Then the specimen cells lie in the region of cells Aor in the region of cells B partitioned by the partition line L, and itbecomes feasible to determine to which cell group the specimen cellsbelong. In (C) of FIG. 8, the result obtained is such that seven cellsout of the thirteen specimen cells are classified in cells A andremaining six cells in cells B. Of course, as shown in (D) and (E) ofFIG. 8, it is also possible to clearly show each data value plotted onthe two-component scatter diagram in association with any one of cellson the quantitative phase image and on the reflection interferenceimage.

The partition line may be provided as a rectangular region parallel tothe vertical and horizontal axes by visual inspection on thetwo-component scatter diagram, or a linear discriminant to distinguishtwo groups may be applied by use of the discriminant analysis techniqueof statistical analysis. When cells are discriminated by a lineardiscriminant, discriminant scores are obtained based on the lineardiscriminant obtained, for individual cells, and then one of two groupstakes positive values of discriminant scores and the other negativevalues, thus permitting simple discrimination of cells.

(Operation and Effects of First Example)

The below will describe the operation and effects of the cellobservation device 1 according to the first example described above. Thecell observation device 1 of the present example is provided with thereflection interference measurement unit consisting of the reflectioninterference measurement light source 106, the reflection interferencedetection camera 110, and the first extraction unit 204, whereby thefirst parameter is obtained based on the reflected light from the cells.The device is also provided with the quantitative phase measurement unitconsisting of the quantitative phase measurement light source 105, thequantitative phase detection camera 112, and the second extraction unit205, whereby the second parameter is obtained based on the transmittedlight from the cells. As described above, the cell observation device 1of the present example is provided with both of the reflectioninterference measurement unit and the quantitative phase measurementunit so as to be able to acquire both of the first parameter and thesecond parameter, which allows the user to obtain the greater amount ofinformation to appropriately determine and evaluate the state of cells.Furthermore, the first parameter is extracted from the reflectioninterference image in alignment with the quantitative phase image andthe second parameter is extracted from the quantitative phase image inalignment with the reflection interference image; therefore, it can besaid that there is consistency achieved between the two parameters as toany part of cells. Namely, the alignment between the two images by theimage alignment unit 201 in the present example makes the two parametersapplicable as parameters to determine and evaluate a state of anidentical portion of cells.

In the present example, the third parameter is further obtained inaddition to the first parameter and the second parameter. Since thisthird parameter is a parameter obtained after matching of cell contoursbetween the quantitative phase image and the reflection interferenceimage, it is different in property from the first parameter and thesecond parameter and when the user further obtains this third parameter,the user has a greater amount of information to appropriately determineand evaluate the state of cells.

There is no user's intervention in the process of generating thereflection interference image after contour application by extractingthe contours of cells from the quantitative phase image and applying thecontours to the reflection interference image. On the other hand, theconventional method with the reflection interference microscope(reflection interference method) requires preliminary recognition ofcontours of individual cells and therefore the reflection interferencemethod is often used in combination with another method for contourrecognition. As described above, Non Patent Literature 1 above disclosesthe combination of the reflection interference method with thefluorescence method, but the operator determines the contours of cellsby handwriting because it was difficult to automatically extract thecell contours. Non Patent Literature 2 discloses the combination of thereflection interference method with the transmission illumination methodbut mentions nothing about automatic determination of cell contourswithout the aid of human hand. Furthermore, Non Patent Literature 3discloses the combination of the reflection interference method with thebright field method and mentions automatic extraction of cell contours,more or less; however, for example, it describes that the user needs tomanually set the optimum threshold for contour extraction while viewingthe bright field image, and thus admits the necessity for user'sintervention to some extent, thus failing to automatically determine thecell contours without the aid of human hand. In contrast to it, in thepresent example the processing including the cell contour extractionfrom the quantitative phase image, the application of the contours tothe reflection interference image, and the generation of the reflectioninterference image after contour application is carried out in theunaided manner by the contour extraction unit 202 and the contourapplication unit 203. The unaided execution of these processesremarkably improves the work efficiency and achieves considerablereduction in operation time in conjunction therewith.

To determine how much the operation time can be reduced byautomatization of the segmentation by the contour extraction unit 202and the contour application unit 203, an experiment was conducted incomparison with the case of Non Patent Literature 3. Approximately ahundred cells existing in the field were photographed in the brightfield (in the case of Non Patent Literature 3) and in the quantitativephase (in the case of the present example), and the time necessary forthe segmentation process was measured for each of them. In the brightfield case, it was difficult to perform the segmentation with athreshold of brightness, and therefore the segmentation was carried outby tracing each of contours of cells with a mouse by an operator's handhaving ordinary work performance. In this case, 150 seconds were neededfor the segmentation of a hundred cells. On the other hand, in the caseof the phase image of cells obtained in quantitative phase, thesegmentation was carried out by the automatic threshold and imageprocessing in the contour extraction unit 202 and the contourapplication unit 203. In this case, a hundred and eight cells weresegmented in an operation processing time of three seconds, therebyachieving the significant time reduction from 150 seconds to 3 seconds.As understood from the above experiment result, when the segmentation iscarried out in the unaided manner with provision of the contourextraction unit 202 and the contour application unit 203 of the presentexample, the work efficiency improves remarkably and the significantreduction in operation time is achieved in conjunction therewith.

In the present example, the information based on the adhesion state ofcells to the substrate is obtained including the information such as theadhesion area of cells to the substrate, the ratio of the adhesion areato the overall imaging range, and the adhesion condition (pattern andtwo-dimensional distribution) of cells to the substrate, and the user isallowed to appropriately determine and evaluate the state of cells,using these pieces of information.

In the present example, the information obtained includes theinformation based on the optical thickness, area, and volume of cells,or the change of refractive index in cells, and the user is allowed toappropriately determine and evaluate the state of cells, using thesepieces of information.

In the present example, the information such as the ratio of theadhesion area to the overall area of cells is obtained as informationbased on the adhesion state to the substrate in the contours of thecells, i.e., in the range of the space occupied by the cells, and theuser is allowed to appropriately determine and evaluate the state ofcells, using this kind of information.

In the present example, the user is allowed to determine the type orstate of cells as an unknown specimen, based on the reference data.

Second Example

The second example of the present invention will be described below. Thesecond example includes all the constituent elements of the firstexample shown in FIG. 1 and is further characterized by the quantitativephase measurement light source 105.

Conventionally, the light source for acquisition of phase changefrequently used was one to emit a laser light beam with high coherency.However, when the laser beam is used, the measurement is often affectedby background noise due to excessive interference originating in anoptical system such as an objective lens or by scattering light noisefrom high-index granules in cells. The quantitative phase measurement inthe cell observation device 1 of the present example is characterized byusing low-coherent light with a wide wavelength band and with lowcoherency, as the illumination light source. This feature can reduce thenoise due to excessive interference and ensure stabler measurement. Inan example, the noise due to excessive interference was reduced by useof an SLD (super luminescent diode) with the center wavelength of 830 nmand the wavelength band of about 20 nm, thus succeeding in stablermeasurement.

Third Example

The third example of the present invention will be described below. Thethird example includes all the constituent elements of the first exampleshown in FIG. 1 and is further characterized by the reflectioninterference measurement light source 106.

Conventionally, the illumination light used for obtaining a sufficientcontrast was illumination light with the use of a band-pass filter tolimit the wavelength band to some extent. However, the band-passedillumination light has high coherency and it was often the case thatinterference fringes are reflected in the image due to reflection frominterfaces between a culture solution and upper cell membranes of cellsunrelated to the adhesion faces of cells. The reflection interferencemeasurement in the cell observation device 1 of the present example useslow-coherent light with a wide wavelength band and with low coherency.The use of the illumination light with the wide wavelength band cannarrow the distance of occurrence of interference and allows thereflection interference image to be taken as being limited to theadhesion faces of the cells to the substrate.

FIG. 9 shows the difference of the reflection interference image withthe use of the illumination light having the wide wavelength band of 420nm to 750 nm from those with the use of the illumination lightband-passed in the narrow wavelength band of about 30 nm around thecenter wavelength of 480 nm or 530 nm. With the illumination lightband-passed in the narrow wavelength band as shown in (A) of FIG. 9 and(B) of FIG. 9, reflection from interfaces between the culture solutionand the upper cell membranes of cells different from the adhesion facesof cells is observed like interference fringes over the image, whereaswith the use of the illumination light having the wide wavelength bandas shown in (C) of FIG. 9, no interference is observed due to reflectedlight from the upper parts of cells and thus it becomes feasible toextract only the information more limited to the adhesion faces ofcells.

In the present example, the light source used is a halogen lamp with awide band of radiation wavelengths and output light therefrom is passedthrough a band-pass filter with an arbitrary wide wavelength band in thevisible-to-near infrared wavelength zone from about 420 nm to about 800nm. The reflection interference image can be extracted as being limitedto the adhesion faces of cells, with the use of the band-passed lighthaving the center wavelength of 500 nm to 1000 nm and the full width athalf maximum of not less than 100 nm. Such devising of illumination isvery effective in acquisition of quantitative parameters from cells.

Fourth Example

The fourth example of the present invention will be described below. Thefourth example includes all the constituent elements of the firstexample shown in FIG. 1 and is further characterized by the illuminationmethod for acquisition of the reflection interference image. FIG. 10 isa schematic diagram of a configuration of a cell observation device 1Aaccording to the fourth example. As shown in FIG. 10, the cellobservation device 1A is further provided with a ring slit 113.

In general, measurement has to be carried out with as many cells aspossible, in order to acquire statistically significant data or todiscover dissimilar cells rarely mixed in a large number of cells. Forthat purpose, it is desirable to measure a wide field at once with theuse of an objective lens having a magnification as low as possible.However, a low-magnification objective lens has a low NA, andillumination light with a low NA includes a lot of components impingingvertically on a sample and causes a phenomenon in which the light isreflected at the interface between air and a solution present above thecells and the cells are illuminated with reflected light. This causes amorphological image of cells unrelated to the adhesion faces, to beincluded on the observation side. Furthermore, the illumination lightpassing through the central region of the objective lens is reflectedinside the objective lens, and the reflected light is included in alarge quantity on the observation side to become high background light,causing reduction in contrast of the reflection interference image ofadhesion faces.

In the reflection interference measurement in the cell observationdevice 1A according to the present example, when a low-NA objective lens104A is used, as shown in FIG. 10, a slit 113 of a ring shape isdisposed at a position conjugate with an aperture stop on the reflectioninterference measurement light source 106 side of the objective lens104A. The illumination light from the reflection interferencemeasurement light source 106 passes through the slit 113 opening in thering shape and thus the illumination light passes through the peripherywithout passing through the center of the objective lens 104A;therefore, the cells 101 are illuminated with the use of only high-NAangled light, which can reduce the influence of reflected light from thesolution above the cells 101. The illumination using the ring-shape slit113 can reduce not only the reflection due to the low-NA objective lens104A, but also generally the background light due to the reflectioninside the objective lens 104A. For allowing the slit 113 opening in thering shape to be changed for each objective lens 104A so as to adapt tothe pupil diameter of the objective lens 104A to be used, a plurality ofring slits 113 adapting to respective objective lenses 104A to be usedare mounted on a disk and the disk is rotated according to needs,thereby achieving a configuration to allow the user to select one of thering slits 113.

FIG. 11 shows the reflection interference image without use of the ringslit 113 and the reflection interference image with use thereof. (A) inFIG. 11 shows the reflection interference image without use of the ringslit 113. The cells are illuminated with reflected light from theinterface between the solution above the cells and air and a cellcontour image is observed over the adhesion image of cells, as indicatedby an arrow. On the other hand, in the case where the illuminationoptical system is provided with the ring slit 113, as shown in (B) ofFIG. 11, illumination components traveling straight toward the interfacebetween the solution above the cells and air are cut so as to preventthe cells from being illuminated with reflected light from the top, andtherefore the information on the adhesion faces of cells can be observedwith good contrast. Such devising of illumination is very effective inacquisition of quantitative parameters from the cells.

Fifth Example

The fifth example of the present invention will be described below. Thefifth example includes all the constituent elements of the first exampleshown in FIG. 1 and is further characterized by the vessel housing thecells 101.

FIG. 12 is a drawing showing the vessel 102A in the fifth example. Oneof features of the vessel 102A is an antireflection coat 114 on the sideopposite to the adhesion faces of cells 101 in the vessel 102A housingthe cells 101 (or on the objective lens side of the observation area ofthe vessel 102A) (which will also be referred to hereinafter as “feature1”). One of the other features of the vessel 102A is provision of anobservation window 116 such as glass parallel to such a height as tocontact the culture solution 115, in order to keep the height of theculture solution 115 constant, in the upper part of the vessel 102A(which will also be referred to hereinafter as “feature 2”). Stillanother feature of the vessel 102A is an antireflection coat 117 on theobservation window 116 outside the vessel 102A (or on the air sideopposite to the face in contact with the culture solution 115) (whichwill also be referred to hereinafter as “feature 3”).

Feature 1 exerts a pronounced effect on acquisition of the reflectioninterference image. For the purpose of measuring the cells 101 as manyas possible, it is preferable to use a low-magnification objective lens.However, the low-magnification objective lens has a low NA, which isusually not of an oil immersion or water immersion type, but isgenerally an objective lens of a dry type. However, when the reflectioninterference image is taken using the objective lens of the dry type,the illumination light emerging from the objective lens is significantlyreflected on the bottom surface of the vessel housing the cells 101.This is because the refractive index difference between air and glass ofthe bottom surface of the vessel is large. For this reason, thebackground light increases considerably and it becomes almost difficultto observe the reflection interference image of the adhesion faces ofcells 101. This is the reason why the objective lens of the oilimmersion or water immersion type has been used heretofore forreflection interference observation.

Therefore, the fifth example enabled the acquisition of the reflectioninterference image with the use of the objective lens of the dry type,by providing the antireflection coat 114 on the side opposite to theadhesion faces of the cells 101 in the vessel 102A housing the cells 101(or on the objective lens side of the observation region of the vessel102A). As shown in FIG. 13, the illumination light from the objectivelens without the antireflection coat 114 is reflected approximately 4%at the interface between air and glass, whereas when the bottom surfaceof glass is subjected to an antireflection coat treatment to control thereflectance R to about 0.5% in the wavelength range of the illuminationlight (420 nm to 720 nm), the background light can be reduced to oneeighth or below. For this reason, the fifth example allows thereflection interference image of the cell adhesion faces to be obtainedwith high contrast, even with the use of the objective lens of the drytype.

FIG. 14 shows the reflection interference image taken with the use ofthe dry objective lens in the configuration where the antireflectioncoat 114 is laid on the bottom surface of the vessel 102A. Without theantireflection coat 114, as shown in (A) of FIG. 14, the reflectioninterference image comes to have extremely low contrast because of thesignificant reflection from the bottom surface of the vessel 102A. Incontrast to it, when the vessel used is the vessel 102A with theantireflection coat 114 on the bottom surface, as shown in (B) of FIG.14, high contrast can be achieved even with the objective lens of thedry type.

Feature 2 is extremely effective in acquisition of the quantitativephase image. In acquisition of the quantitative phase image, variationin height of the liquid surface due to vibration of the solution leadsto variation in optical path length, so as to produce noise inmeasurement. For this reason, measurement is preferably carried outwhile keeping the height of the liquid surface unchanged. In performingobservation without interruption, however, there are cases where amedical fluid is dispensed in the middle and a change thereafter ismeasured, and in such cases the dispensation of medical fluid inevitablycauses vibration of the liquid surface. Therefore, the fifth example ischaracterized by the top part (or lid) of the vessel 102A housing thecells 101.

Namely, the upper part (or lid) of the vessel 102A is provided with theobservation window 116 such as glass parallel to such a height as tocontact the culture solution 115, so as to keep the height of theculture solution 115 constant. Thanks to the existence of theobservation window 116, the height of the solution in the observationregion of the vessel 102A housing the cells 101 is kept constant betweenthe bottom surface of the vessel 102A as an observation surface incontact with the cells 101 and the observation window 116 in the upperpart of the vessel 102A. The observation window 116 is provided in therange corresponding to the observation region of the bottom surface ofthe vessel 102A and an aperture is formed in the periphery to allow thedispensation of medical fluid. In acquisition of the quantitative phaseimage, the observation window 116 is preferably parallel to the bottomsurface of the sample. When the observation window 116 to keep theheight of the culture solution 115 constant is provided in contact withthe culture solution 115 in the upper part of the vessel 102A, thequantitative phase image is stable in terms of time and the measurementresult of optical path lengths of cells 101 can be always obtained underthe stable condition. The observation window 116 in the upper part ofthe vessel 102A also serves as a lid of the vessel 102A and it isfeasible to maintain the inside in an aseptic condition with the lidclosed. With consideration to the dispensation of medical fluid, the lidpart may be provided with an aperture for the dispensation of medicalfluid.

Feature 3 is provision of the antireflection coat 117 on the observationwindow 116 outside the vessel 102A (or on the air side opposite to thesurface in contact with the culture solution 115), for acquisition ofthe reflection interference image. Without the antireflection coat 117,the difference is large between the refractive indices of the glass ofthe observation window 116 in contact with the solution above the cells101 and the exterior air and the illumination light from the objectivelens for the purpose of acquisition of the reflection interference imageis reflected approximately 4% on the observation window 116. Thereflected light illuminates the cells 101 to project a contour image ofcells 101 onto the reflection interference image. This is informationobstructive to extraction of the information limited to the adhesionfaces from the reflection interference image and inhibits acquisition ofa clear reflection interference image. In the fifth example, therefore,the antireflection coat to control the reflectance to about 0.5% in thewavelength range of the illumination light is provided on theobservation window 116 outside the vessel 102A, whereby the reflectancecan be reduced to about ⅛. This suppresses the influence of reflectedlight from the top surface, whereby the reflection interference image ofthe cell adhesion faces can be obtained with high contrast.

FIG. 15 shows an improvement of the reflection interference image in thecase where the antireflection coat 117 is provided on the observationwindow 116 in the upper part of the vessel 102A. Without the treatment,as shown in (A) of FIG. 15, the reflected light from the interfacebetween the observation window 116 in the upper part of the vessel 102Aand air illuminates the cells 101 from the top to project a contourimage of cells 101 onto the image. On the other hand, when theantireflection coat 117 is provided on the air side of the observationwindow 116 in the upper part, as shown in (B) of FIG. 15, the reflectionfrom the top is reduced, whereby the reflection interference image ofthe adhesion faces of cells 101 can be observed with good contrast.

If stationary-standing observation is carried out, for example, in astate in which no medical fluid or the like is dispensed duringmeasurement, so as to prevent disturbance of the culture solution 115 inthe vessel 102A housing the cells 101, the provision of the foregoingobservation window 116 is not essential, and the vessel may be vessel102B having only feature 1 with the antireflection coat 114 on thebottom surface of the vessel, as shown in FIG. 16.

Sixth Example

The sixth example of the present invention will be described below. Thesixth example is different in the image acquisition unit 10, out of theconstituent elements of the first example shown in FIG. 1. Namely, thepresent example is characterized by the configuration of the opticalsystem for simultaneously carrying out the quantitative phasemeasurement and the reflection interference measurement.

FIG. 17 shows an image acquisition unit 10A in the sixth example. Whencompared with the image acquisition unit 10 in the first example, theimage acquisition unit 10A is configured without use of the totalreflection mirror 109. Namely, the quantitative phase optical systemconsists of only the diffractive interference optical system 111. Thisconfiguration can substantialize the simple configuration and providesthe advantage of being easy to construct the device.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 installed abovethe vessel 102 housing the cells 101 travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Thenthe illumination light travels through the half mirror 107 and thenthrough the dichroic mirror 108 for separation of the quantitative phaseimage and the reflection interference image by wavelengths, to form aninterference image between object light and reference light in thediffractive interference optical system 111 for phase measurement, andthe interference fringe image is taken by the quantitative phasedetection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isreflected by the half mirror 107, then travels through the objectivelens 104, and is incident into the vessel 102 housing the cells 101 as ameasurement target, from the bottom side. Reflected light from theadhesion faces of cells 101 on the bottom surface of the vessel 102causes interference according to the adhesion distances of the cells101, the resultant reflection interference light is condensed by theobjective lens 104, the light travels through the half mirror 107, andonly the reflection interference image is reflected by the dichroicmirror 108 for separation of the quantitative phase image and thereflection interference image by wavelengths, to be picked up by thereflection interference detection camera 110.

Seventh Example

The seventh example of the present invention will be described below.The seventh example is different in the image acquisition unit 10, outof the constituent elements of the first example shown in FIG. 1.Namely, the present example is characterized by the configuration of theoptical system for simultaneously performing the quantitative phasemeasurement and the reflection interference measurement.

FIG. 18 shows an image acquisition unit 10B in the seventh example. Whencompared with the image acquisition unit 10 in the first example, theimage acquisition unit 10B is configured without use of the totalreflection mirror 109. Therefore, the present example can realize thesimple configuration and has the advantage of being easy to constructthe device. Since the half mirror 107 of the reflection interferenceincidence optical system is not a common optical system, thequantitative phase image is taken without passage through the halfmirror 107, thereby providing the advantage of reduction in lightquantity loss on the quantitative phase side.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 installed abovethe vessel 102 housing the cells 101 travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Then,without passing through the half mirror 107, the illumination lighttravels through the dichroic mirror 108 for separation of thequantitative phase image and the reflection interference image bywavelengths, to form the interference image between object light andreference light in the diffractive interference optical system 111 forphase measurement, and the interference fringe image is taken by thequantitative phase detection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isreflected by the half mirror 107 and the dichroic mirror 108, thentravels through the objective lens 104, and is incident into the vessel102 housing the cells 101 as a measurement target, from the bottom side.Reflected light from the adhesion faces of the cells 101 on the bottomsurface of the vessel 102 causes interference according to the adhesiondistances of the cells 101, the resultant reflection interference lightis condensed by the objective lens 104, and then the light travels againvia the dichroic mirror 108 and the half mirror 107 to be picked up bythe reflection interference detection camera 110.

Eighth Example

The eighth example of the present invention will be described below. Theeighth example is different in the image acquisition unit 10, out of theconstituent elements of the first example shown in FIG. 1. Namely, thepresent example is characterized by the configuration of the opticalsystem for simultaneously performing the quantitative phase measurementand the reflection interference measurement.

FIG. 19 shows an image acquisition unit 10C in the eighth example. Whencompared with the image acquisition unit 10 in the first example, theimage acquisition unit 10C is configured without use of the totalreflection mirror 109. Namely, the quantitative phase optical systemconsists of only the diffractive interference optical system 111, thusrealizing the simple configuration and providing the advantage of beingeasy to construct the device. Since the half mirror 107 of thereflection interference incidence optical system is not a common opticalsystem, the quantitative phase image is taken without passing throughthe half mirror 107, thereby providing the advantage of reduction inlight quantity loss on the quantitative phase side.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 installed abovethe vessel 102 housing the cells 101 travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Then,without passing through the half mirror 107, the illumination light isreflected from the dichroic mirror 108 for separation of thequantitative phase image and the reflection interference image bywavelengths, to form the interference image between object light andreference light in the diffractive interference optical system 111 forphase measurement, and the interference fringe image is taken by thequantitative phase detection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isreflected via the half mirror 107 and the dichroic mirror 108, travelsthrough the objective lens 104, and is then incident into the vessel 102housing the cells 101 as a measurement target, from the bottom side.Reflected light from the adhesion faces of the cells 101 on the bottomsurface of the vessel 102 causes interference according to the adhesiondistances of the cells 101, the resultant reflection interference lightis condensed by the objective lens 104, and then the light travels againvia the dichroic mirror 108 and the half mirror 107 to be picked up bythe reflection interference detection camera 110.

Ninth Example

The ninth example of the present invention will be described below. Theninth example is different in the image acquisition unit 10, out of theconstituent elements of the first example shown in FIG. 1. Namely, thepresent example is characterized by the configuration of the opticalsystem for simultaneously performing the quantitative phase measurementand the reflection interference measurement.

FIG. 20 shows an image acquisition unit 10D in the ninth example. Whencompared with the image acquisition unit 10 in the first example, theimage acquisition unit 10D is configured without use of the totalreflection mirror 109 and the dichroic mirror 108, and has one commonlight source 105D instead of the quantitative phase measurement lightsource 105 and the reflection interference measurement light source 106.Therefore, the present example can realize the simple configuration andprovides the advantage of being easy to construct the device. However,the present example needs to have a second objective lens 104D.

In the quantitative phase measurement, the illumination light emittedfrom the common light source 105D is reflected by the half mirror 107,then travels through the objective lens 104, passes through the vessel102 housing the cells 101, and is condensed by the second objective lens104D. Then the interference image between object light and referencelight is formed in the diffractive interference optical system 111 andthe interference fringe image is taken by the quantitative phasedetection camera 112.

In the reflection interference measurement, the illumination lightemitted from the common light source 105D is reflected by the halfmirror 107, travels through the objective lens 104, and is incident intothe vessel 102 housing the cells 101 as a measurement target, from thebottom side. The reflected light from the adhesion faces of the cells101 on the bottom surface of the vessel 102 causes interferenceaccording to the adhesion distances of the cells 101, the resultantreflection interference light is condensed by the objective lens 104,and the light travels again via the half mirror 107 to be picked up bythe reflection interference detection camera 110.

Tenth Example

The tenth example of the present invention will be described below. Thetenth example is different in the image acquisition unit 10, out of theconstituent elements of the first example shown in FIG. 1. Namely, thepresent example is characterized by the configuration of the opticalsystem for simultaneously performing the quantitative phase measurementand the reflection interference measurement.

FIG. 21 shows an image acquisition unit 10E in the tenth example. Whencompared with the image acquisition unit 10 in the first example, theimage acquisition unit 10E is configured without use of the totalreflection mirror 109, the half mirror 107, and the dichroic mirror 108and, the light beam for quantitative phase measurement and the lightbeam for reflection interference measurement are separated by anglesinstead of wavelengths. The present example needs to have a secondobjective lens 104E.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 installed abovethe vessel 102 housing the cells 101 travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Then,without passing via the half mirror 107 and the dichroic mirror 108, theinterference image between object light and reference light is formed inthe diffractive interference optical system 111 and the interferencefringe image is taken by the quantitative phase detection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isobliquely incident into the vessel 102 housing the cells 101 as ameasurement target, from the bottom side. The reflected light from theadhesion faces of the cells 101 on the bottom surface of the vessel 102causes interference according to the adhesion distances of the cells101, the resultant reflection interference light travels again obliquelythrough the second objective lens 104E to be condensed thereby, and thenthe light travels without passing via the half mirror 107 or thedichroic mirror 108, to be picked up by the reflection interferencedetection camera 110. The present example requires adjustment of anglesamong the reflection interference measurement light source 106, thesecond objective lens 104E, and the reflection interference detectioncamera 110, but can be configured without use of the total reflectionmirror 109, half mirror 107, and dichroic mirror 108, thus providing theadvantage of implementation of the simple configuration.

Eleventh Example

The eleventh example of the present invention will be described below.The eleventh example is different in the image acquisition unit 10, outof the constituent elements of the first example shown in FIG. 1.Namely, the present example is characterized by the configuration of theoptical system for simultaneously performing the quantitative phasemeasurement and the reflection interference measurement.

FIG. 22 shows an image acquisition unit 10F in the eleventh example.When compared with the image acquisition unit 10 in the first example,the image acquisition unit 10F is configured without use of the totalreflection mirror 109, the half mirror 107, and the dichroic mirror 108and, the light beam for quantitative phase measurement and the lightbeam for reflection interference measurement are separated by anglesinstead of wavelengths. The present example needs to have a secondobjective lens 104F.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 is incidentinto the vessel 102 housing the cells 101 as a measurement target, fromthe bottom side. The illumination light travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Then,without passing via the total reflection mirror 109, the half mirror107, or the dichroic mirror 108, the interference image between objectlight and reference light is formed in the diffractive interferenceoptical system 111 and the interference fringe image is taken by thequantitative phase detection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isobliquely incident into the vessel 102 housing the cells 101 as ameasurement target, from the bottom side. The reflected light from theadhesion faces of the cells 101 on the bottom surface of the vessel 102causes interference according to the adhesion distances of the cells101, the resultant reflection interference light travels again obliquelythrough the second objective lens 104F to be condensed thereby, and thelight travels without passing via the total reflection mirror 109, thehalf mirror 107, or the dichroic mirror 108, to be picked up by thereflection interference detection camera 110. The present examplerequires adjustment of angles among the reflection interferencemeasurement light source 106, the second objective lens 104F, and thereflection interference detection camera 110, but can be configuredwithout use of the total reflection mirror 109, the half mirror 107, andthe dichroic mirror 108, thereby providing the advantage ofimplementation of the simple configuration.

Twelfth Example

The twelfth example of the present invention will be described below.The twelfth example is different in the image acquisition unit 10, outof the constituent elements of the first example shown in FIG. 1.Namely, the present example is characterized by the configuration of theoptical system for simultaneously performing the quantitative phasemeasurement and the reflection interference measurement and,specifically, the quantitative phase measurement method is modified intoa two-beam system.

FIG. 23 shows an image acquisition unit 10G in the twelfth example. Whencompared with the image acquisition unit 10A in the sixth example, theimage acquisition unit 10G is different in that, without use of thediffractive interference optical system 111, illumination and referencelight beams from the quantitative phase measurement light source 105 areguided through respective optical paths 118G and 119G to thequantitative phase detection camera 112. The present example providesthe advantage of being capable of constructing the device without thediffractive interference optical system 111. The present example isconfigured by adopting the two-beam system, instead of use of thediffractive interference optical system 111, as the quantitative phasemeasurement method, so as to produce the reference light beam throughthe separate optical path, and finally making the object beam and thereference beam interfere with each other.

In the quantitative phase measurement, the illumination light from thequantitative phase measurement light source 105 travels through theoptical path 118G for illumination light, then travels through a lens120G installed above the vessel 102, passes through the cells 101 in thevessel 102, and is condensed by the objective lens 104. Then the lighttravels via the half mirror 107, passes through the dichroic mirror 108,and further travels via a half mirror 121G and a lens 122G to reach thequantitative phase detection camera 112. On the other hand, thereference beam from the quantitative phase measurement light source 105travels through the optical path 119G for the reference beam, and thentravels in order through a lens 123G, the half mirror 121G, and the lens122G to reach the quantitative phase detection camera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isreflected by the half mirror 107, travels through the objective lens104, and is incident into the vessel 102 housing the cells 101 as ameasurement target, from the bottom side. The reflected light from theadhesion faces of the cells 101 on the bottom surface of the vessel 102causes interference according to the adhesion distances of the cells101, the resultant reflection interference light is condensed again bythe objective lens 104, and travels via the half mirror 107, and onlythe reflection interference image is reflected by the dichroic mirror108 for separation of the quantitative phase image and the reflectioninterference image by wavelengths, to be picked up by the reflectioninterference detection camera 110.

Thirteenth Example

The thirteenth example of the present invention will be described below.The thirteenth example is different in the image acquisition unit 10,out of the constituent elements of the first example shown in FIG. 1.Namely, the present example is characterized by the configuration of theoptical system for simultaneously performing the quantitative phasemeasurement and the reflection interference measurement.

FIG. 24 shows an image acquisition unit 10H in the thirteenth example.When compared with the image acquisition unit 10A in the sixth example,the image acquisition unit 10H is different in that it is furtherprovided with an illumination wavelength switch device 124H and anobservation wavelength switch device 125H. The illumination wavelengthswitch device 124H is a device that switches the wavelengths of theillumination light from the reflection interference measurement lightsource 106 to other wavelengths. The observation wavelength switchdevice 125H is a device that switches the wavelengths of the observationlight from the dichroic mirror 108 to other wavelengths. Since the imageacquisition unit 10H is provided with these devices, it is able toacquire a desired fluorescence image or the like by the reflectioninterference detection camera 110. FIG. 24 shows the illuminationwavelength switch device 124H and the observation wavelength switchdevice 125H configured to perform switching of wavelengths by rotation,as an example.

In the quantitative phase measurement, the illumination light emittedfrom the quantitative phase measurement light source 105 installed abovethe vessel 102 housing the cells 101, travels through the vessel 102housing the cells 101, to be condensed by the objective lens 104. Thenthe light travels via the half mirror 107, passes through the dichroicmirror 108 for separation of the quantitative phase image and thereflection interference image by wavelengths, to form the interferenceimage between object light and reference light in the diffractiveinterference optical system 111 for phase measurement, and theinterference fringe image is taken by the quantitative phase detectioncamera 112.

In the reflection interference measurement, the illumination lightemitted from the reflection interference measurement light source 106 isswitched to desired wavelengths by the illumination wavelength switchdevice 124H, and the light is reflected by the half mirror 107, travelsthrough the objective lens 104, and is then incident into the vessel 102housing the cells 101 as a measurement target, from the bottom side. Thereflected light from the adhesion faces of the cells 101 on the bottomsurface of the vessel 102 causes interference according to the adhesiondistances of the cells 101, the resultant reflection interference lightis condensed by the objective lens 104, the light travels again via thehalf mirror 107, only the reflection interference image is reflected bythe dichroic mirror 108, and the reflected light is switched intodesired wavelengths by the observation wavelength switch device 125H, tobe picked up by the reflection interference detection camera 110.

Fourteenth Example

The fourteenth example of the present invention will be described below.The fourteenth example is different in the half mirror 107, out of theconstituent elements of the first example shown in FIG. 1. Namely, thefourteenth example includes all the constituent elements of the firstembodiment, of which the characteristics of the half mirror 107 arefurther improved.

An ordinary half mirror generally has an equal separation ratiothroughout the entire wavelength zone. If such a half mirror is used asthe half mirror 107 in the present embodiment, the transmittance of thequantitative phase image will be limited by the half mirror 107 when thequantitative phase image passes through the half mirror 107 placed inthe optical path for the purpose of reflection interference. Then thefourteenth example employs a half mirror 107A which is located betweenthe reflection interference measurement light source 106 and the camera110 and between the quantitative phase measurement light source 105 andthe camera 110 and which can have different ratios of reflection totransmittance depending upon wavelengths, as shown in an imageacquisition unit 10I in FIG. 25. The fourteenth example is based on thepremise that the quantitative phase measurement light source 105 and thereflection interference measurement light source 106 emit theirrespective illumination beams of different wavelengths, so as to avoidan overlap between the wavelengths of the illumination beams forreflection interference and quantitative phase. The wavelengthcharacteristics of the half mirror 107A are such wavelengthcharacteristics that it works as a half mirror(transmission:reflection=80:20 in FIG. 25) in the observation wavelengthregion of reflection interference and it transmits maximum light in theobservation wavelength region of quantitative phase. This configurationdecreases a loss in light quantity of the observation light forquantitative phase.

For example, in a case where the visible light (420 nm to 750 nm) isused in reflection interference and light with the center wavelength of830 nm and the full width at half maximum of 20 nm is used inquantitative phase, the wavelength characteristics of the half mirror107A are set so that the ratio of reflection:transmission is set at20:80 for the visible light (420 nm to 750 nm) and the ratio ofreflection:transmission is set at 5:95 for 800 nm and above. This setupallows 80% of observation light to be obtained for the reflectioninterference and allows a signal to be obtained at the transmittance of95% for quantitative phase, independent of the transmittance in thereflection interference of the half mirror 107A.

Fifteenth Example

The fifteenth example of the present invention will be described below.The fifteenth example has the same configuration as the configuration ofthe first example shown in FIG. 1, but may be configured without thereference storage unit 208 in FIG. 1, though not shown, because theanalysis unit 207 performs the processing without depending on thereference storage unit 208.

In the fifteenth example, the analysis unit 207 determines a type orstate of a cell as an unknown specimen, independently of the referencedata. Namely, the analysis unit 207 selects predetermined parametersfrom the parameters extracted for the unknown cell and determines thetype or state of unknown cell, using the predetermined parameters thusselected. The analysis unit 207 may be configured so as to automaticallyselect parameters indicative of peculiar values as the predeterminedparameters. A separate processing means may be provided in order toperform the parameter selection process. On the other hand, the analysisunit 207 may be configured to select the predetermined parameters, basedon an input from the user.

FIG. 26 shows a screen 40 displayed for the user, in the configurationwherein the analysis unit 207 is configured to select the predeterminedparameters, based on the input from the user, in the fifteenth example.A screen field 40A is a picture that shows kinds of parameters for theuser and that is provided for accepting selection by the user. In theexample of the screen field 40A, the user selects the inertia andkurtosis as the predetermined parameters. The user may be allowed to usea selection tool 40A1 or the like to select other parameters notdisplayed on the screen field 40A. A screen field 40B shows an exampleof display on a graph reflecting the parameters selected by the user.After the user checks the screen field 40B, the user may return to thescreen field 40A to select other parameters, or may advance directly toa screen field 40C. The screen field 40C is a picture displaying animage of the cell.

In the fifteenth example of this configuration, the analysis unit 207can perform the processing, without depending upon the reference data.Therefore, the reference storage unit 207 may be omitted, whichsimplifies the device configuration. This mode of embodiment is suitablyapplicable, for example, to the case where it is known in advance thatthe cells in the sample are separated into myeloid stem cells andosteoclasts and where the cells are to be discriminated from each other.

Sixteenth Example

The sixteenth example of the present invention will be described below.The sixteenth example is different mainly in the operation of theanalysis unit 207, out of the constituent elements of the first exampleshown in FIG. 1. The sixteenth example is based on the premise thatthree or more parameters are extracted by each of the first extractionunit 204, the second extraction unit 205, and the third extraction unit206, or by a combination thereof, and in this case the analysis unit 207performs a principal component analysis on the three or more parametersto determine a type or state of an unknown cell.

The operation in the sixteenth example is briefly summarized as follows.First, three or more parameters are extracted by each of the firstextraction unit 204, the second extraction unit 205, and the thirdextraction unit 206, or by a combination thereof and are output to theanalysis unit 207 (hereinafter “step 1”). Next, the analysis unit 207standardizes the three or more parameters (hereinafter “step 2”). Next,the analysis unit 207 determines a first principal component and asecond principal component, based on the standardized data (hereinafter“step 3”). Then the analysis unit 207 plots cells on a two-componentscatter diagram of the first principal component and the secondprincipal component and performs cell determination (hereinafter “step4”). Each of the steps will be detailed below based on an actualexperiment example.

(Step 1: Parameter Extraction)

In this experiment, four types of cells, each type including ten cells,were used as reference and three unknown cells were discriminated basedthereon. The experiment was conducted based on the assumption that thethree unknown cells were included in any one of the four types of cells,and the purpose of the experiment is to discriminate the three unknowncells as to in which they are included among the four types of cells.The below provides the names, notations, and numbers (n) of therespective cells used as reference, in order.

Rat pancreatic β cell line INS-1 (n=10)

Mouse pancreatic β cell line MIN-6 (n=10)

Human pancreatic cancer cell line MIAPaCa-2 (n=10)

Human uterine cervix cancer cell line HeLa (n=10)

FIG. 27 shows the reflection interference images and quantitative phaseimages of the respective cells used as reference. (A) and (B) in FIG. 27show the reflection interference image and quantitative phase image ofrat pancreatic β cell line (INS-1) in order. (C) and (D) in FIG. 27 showthe reflection interference image and quantitative phase image of mousepancreatic β cell line (MIN-6) in order. (E) and (F) in FIG. 27 show thereflection interference image and quantitative phase image of humanpancreatic cancer cell line (MIAPaCa-2) in order. (G) and (H) in FIG. 27show the reflection interference image and quantitative phase image ofhuman uterine cervix cancer cell line (HeLa) in order.

In this experiment, each of the first extraction unit 204, the secondextraction unit 205, and the third extraction unit 206, or a combinationthereof extracted seven parameters below from the reflectioninterference images and quantitative phase images shown in FIG. 27. Theparameters (1) and (2) below are parameters obtained from thequantitative phase images and the parameters (3) to (7) parametersobtained from the reflection interference images. FIG. 28 shows actuallymeasured values of the seven parameters.

(1) Area of cell

(2) Thickness of cell

(3) Texture of adhesion face: co-occurrence matrix/local uniformity

(4) Texture of adhesion face: co-occurrence matrix/inertia

(5) Contrast of adhesion face: gray level histogram/skewness

(6) Contrast of adhesion face: gray level histogram/kurtosis

(7) Contrast of adhesion face: gray level histogram/(variance)/average

(Step 2: Standardization)

For obtaining components indicative of summarized features (principalcomponents) from the plurality of parameters extracted in above step 1,it is necessary to create a linear formula including the parameters.However, the measured values shown in FIG. 28 are represented bydifferent units depending on the parameters, and the magnitudes of takenvalues are different from each other. If these values are handled asthey are, information will be biased to parameters of large values andit will be difficult to equally extract information from each of theparameters. Then an amount of information of each parameter wascalculated in order to equally extract information from each parameter.As an information amount, the sum of squared deviations from apopulation was calculated from the measured values shown in FIG. 28 andthen the calculation result as shown in FIG. 29 was obtained. However,there were large differences of information amounts among theparameters, as shown in FIG. 29. Since it is desirable to equalize theinformation amounts of the respective parameters, the present experimentadopted standardization of data, based on formula (1) below, for each ofthe parameters.

X′=(Xi−X)/Xsd  (1)

In formula (1), X′ is data after standardization, Xi each measured valueshown in FIG. 28, X an average value in Xi, and Xsd a standard deviationin Xi.

FIG. 30 shows values of the respective parameters after thestandardization. As shown in FIG. 30, as the result of standardization,the information amounts of the respective parameters are equalindependent of the types of parameters. When such data afterstandardization is used, the data can be handled equally.

(Step 3: Calculation of Principal Components)

Next, the analysis unit 207 obtains the first principal component f andthe second principal component g, based on the data after thestandardization in FIG. 30. First, let the standardized data be area=X1,optical thickness=X2, . . . , Π (variance)/average=X7, respectively;then f and g as the two principal components indicative of integratedfeatures are calculated based on formulas (2) and (3) below. In theformulas (2) and (3), a1 to a7 and b1 to b7 are coefficients.

f=a1*X1+a2*X2+ . . . +a7*X7  (2)

g=b1*X1+b2*X2+ . . . +b7*X7  (3)

At this time, the following conditions are set for the coefficients.

a1² +a2² + . . . +a7²=1  (4)

b1² +b2² + . . . +b7²=1  (5)

The reason for setting of the conditions represented by formulas (4) and(5) is that the coefficients a1 to a7, b1 to b7 to maximize thevariances of the principal components f, g are calculated in theprocedure below and thus the coefficients increase without limit unlessthe coefficients are limited by the conditions of formulas (4) and (5).“a1²+a2²+ . . . +a7²” and “b1²+b2²+ . . . +b7²” correspond to themagnitudes of vectors of the coefficients, and when the magnitudes ofthe vectors are set to 1, the first principal component f and the secondprincipal component g as new determination axes can be obtained withoutchange in the information amounts of original data.

For making f and g be uncorrelated straight lines orthogonal to eachother, the condition below is further set.

a1*b1+a2*b2+ . . . +a7*b7=0  (6)

For making the linear equation reflect the information amounts oforiginal data for f as much as possible, it is conceivable to maximizedispersion of values of the component given by f. Since the dispersioncan be considered to be variance, the variance of f is made maximum.When f1, f2, f3, . . . , and f43 represent values of f of the respectivecells with the number of samples of 43, the variance Vf of f is given byformula (7) below and f satisfying the condition that the variance Vfbecomes maximum is calculated.

$\begin{matrix}\left\lbrack {{Math}\mspace{14mu} 1} \right\rbrack & \; \\{{Vf} = \frac{\left( {f_{1} - \overset{\_}{f}} \right)^{2} + \left( {f_{2} - \overset{\_}{f}} \right)^{2} + \left( {f_{3} - \overset{\_}{f}} \right)^{2} + \ldots + \left( {f_{43} - \overset{\_}{f}} \right)^{2}}{43}} & (7)\end{matrix}$

In the formula (7) above,

f   [Math 2]

is the average of f

By the above method, the coefficients a1, a2, . . . , a7 satisfying thecondition of above formula (4) and satisfying the condition of aboveformula (7) are calculated for the first principal component f. FIG. 31shows the coefficients of the principal component f calculated based onthe calculation method described above.

Next, for the second principal component g, the coefficients b1, b2, . .. , b7 are calculated so as to satisfy the condition of above formula(5), satisfy the condition of the same spirit as above formula (7)(i.e., the condition to maximize variance), and further satisfy thecondition of above formula (6). FIG. 32 shows the coefficients of thesecond principal component g calculated based on the calculation methoddescribed above.

Next, the values of f and g are determined for each cell and they aredefined as the first principal component and the second principalcomponent, respectively. FIG. 33 shows the result of calculation of thefirst principal component f and the second principal component g foreach of the cells. (A) in FIG. 33 shows the first principal component fand the second principal component g of the rat pancreatic β cell line(INS-1) and the mouse pancreatic β cell line (MIN-6), and (B) in FIG. 33shows the first principal component f and the second principal componentg of the human pancreatic cancer cell line (MIAPaCa-2) and the humanuterine cervix cancer cell line (HeLa).

(Step 4: Plot and Cell Determination)

The analysis unit 207 plots each cell on the two-component scatterdiagram, using the values of the first principal component f and thesecond principal component g shown in FIG. 33, and performs celldetermination. FIG. 34 shows the result of the plot, which is an examplein which each cell was plotted with the first principal component f onthe horizontal axis and the second principal component g on the verticalaxis. As seen from the plot result of FIG. 34, the scatter diagram ofthe first principal component f and the second principal component g(principal component scatter diagram) shows that the four types of cellgroups form groups that can be readily discriminated according to theirfeature values. This indicates that the features of the respective cellscan be clearly shown by the calculation to maximize the variance of eachprincipal component (above formula (7)). As a result, it was determinedthat the unknown cells (unknown represented by ∘) on the principalcomponent scatter diagram belonged to the group of MIA-PaCa cellsrepresented by Δ. Since the unknown cells were actually MIA-PaCa, it wasconfirmed that the cell determination by this experiment was correct.

Example 16 described above is suitably applicable, particularly, tocases where all of many parameters extracted or some of three or moreextracted parameters are desired to be effectively used for the celldetermination. For example, it is particularly suitable for cases wherethere are a large number of types of cells to be determined and they areto be determined with high accuracy using the large number ofparameters, cases where it is not easy to select two parameters from thelarge number of extracted parameters, and so on. In Example 16, theprincipal component analysis is performed on the large number ofextracted parameters, whereby the large number of parameters can be madeto appropriately affect the cell determination. It is because theprincipal component analysis uses the two principal components properlyreflecting all of the large number of parameters or some of three ormore parameters, instead of selecting two out of the large number ofextracted parameters, to perform the cell determination.

In the description above, the case where the number of parameters was 7was described as an example, but, without having to be limited to it,the present example can be applied to cases of three or more parameters.Although description is omitted in order to avoid redundancy, the sameexperiment as the above experiment was actually conducted with tenparameters and the result obtained was the same as in the case usingseven parameters.

LIST OF REFERENCE SIGNS

-   -   1, 1A, cell observation device; 10, 10A, 10B, 10C, 10D, 10E,        10F, 10G, 10H image acquisition unit; 20 processing unit; 30        sample; 31 grid; 40, 40A, 40B, 40C screen; 101 cells; 102, 102A,        102B vessel; 103 culture space; 104, 104A, 104D, 104E, 104F        objective lens; 105 quantitative phase measurement light source;        105A illumination stop unit; 105D common light source; 106        reflection interference measurement light source; 107, 107A half        mirror; 108 dichroic mirror; 109 total reflection mirror; 110        reflection interference detection camera; 111 diffractive        interference optical system; 112 quantitative phase detection        camera; 113 ring slit; 114 antireflection coat; 115 culture        solution; 116 observation window; 117 antireflection coat; 118G        optical path for illumination light in quantitative phase        measurement; 119G optical path for reference light in        quantitative phase measurement; 120G, 122G, 123G lenses; 121G        half mirror; 124H illumination wavelength switch device; 125H        observation wavelength switch device; 201 image alignment unit;        202 contour extraction unit; 203 contour application unit; 204        first extraction unit; 205 second extraction unit; 206 third        extraction unit; 207 analysis unit; 208 reference storage unit.

INDUSTRIAL APPLICABILITY

The present invention provides the cell observation device and the cellobservation method capable of obtaining the greater amount ofinformation for appropriate determination and evaluation of the cellstate.

1. A cell observation device comprising: a reflection interferencemeasurement light source; a quantitative phase measurement light source;reflection interference imaging unit which images light emitted from thereflection interference measurement light source and reflected from acell, to generate a reflection interference image; quantitative phaseimaging unit which images light emitted from the quantitative phasemeasurement light source and transmitted by the cell, to generate aquantitative phase image; image alignment unit which matches a spatialposition of the reflection interference image with a spatial position ofthe quantitative phase image, thereby to implement alignment between thetwo images; first extraction unit which extracts a first parameter fromthe reflection interference image in the alignment with the quantitativephase image by the image alignment unit; and second extraction unitwhich extracts a second parameter from the quantitative phase image inthe alignment with the reflection interference image by the imagealignment unit.
 2. The cell observation device according to claim 1,further comprising: contour extraction unit which extracts a contour ofthe cell, based on the quantitative phase image; contour applicationunit which applies the contour extracted by the contour extraction unit,to the reflection interference image to generate a reflectioninterference image after contour application; and third extraction unitwhich extracts a third parameter from the reflection interference imageafter contour application.
 3. The cell observation device according toclaim 1, wherein the first parameter is information based on an adhesionstate between a substrate on which the cell is laid, and the cell. 4.The cell observation device according to claim 1, wherein the secondparameter is information based on an optical thickness, area, volume ofthe cell, or a change in refractive index in the cell.
 5. The cellobservation device according to claim 2, wherein the third parameter isinformation based on the adhesion state to the substrate, in the contourof the cell.
 6. The cell observation device according to claim 1,further comprising: reference storage unit which stores as referencedata a parameter preliminarily extracted for the cell of a known type orstate; and analysis unit which determines a type or state of an unknowncell, based on the reference data.
 7. The cell observation deviceaccording to claim 1, further comprising: analysis unit which selects apredetermined parameter from parameters extracted for an unknown celland which determines a type or state of the unknown cell, using thepredetermined parameter selected.
 8. The cell observation deviceaccording to claim 1, further comprising: analysis unit which, when thefirst extraction unit or the second extraction unit extracts three ormore parameters, performs a principal component analysis on said threeor more parameters, thereby to determine a type or state of an unknowncell.
 9. The cell observation device according to claim 1, whereinlow-coherent light is used as the quantitative phase measurement lightsource.
 10. The cell observation device according to claim 1, whereinlow-coherent light is used as the reflection interference measurementlight source.
 11. The cell observation device according to claim 1,further comprising: an objective lens which condenses the light emittedfrom the reflection interference measurement light source and reflectedfrom the cell; and a slit of a ring shape at a position conjugate withan aperture stop on the reflection interference measurement light sourceside of the objective lens.
 12. The cell observation device according toclaim 1, further comprising: a vessel which houses the cell, wherein anantireflection coat is laid on the side of the vessel opposite to anadhesion face of the cell.
 13. The cell observation device according toclaim 1, further comprising: a mirror located between the reflectioninterference measurement light source and the reflection interferenceimaging unit and between the quantitative phase measurement light sourceand the reflection interference imaging unit and having variable ratiosof reflection to transmission depending upon wavelengths.
 14. A cellobservation method comprising: a reflection interference imaging stepwherein reflection interference imaging unit images light emitted from areflection interference measurement light source and reflected from acell, to generate a reflection interference image; a quantitative phaseimaging step wherein quantitative phase imaging unit images lightemitted from a quantitative phase measurement light source andtransmitted by the cell, to generate a quantitative phase image; animage alignment step wherein image alignment unit matches a spatialposition of the reflection interference image with a spatial position ofthe quantitative phase image, thereby to implement alignment between thetwo images; a first extraction step wherein first extraction unitextracts a first parameter from the reflection interference image in thealignment with the quantitative phase image by the image alignment unit;and a second extraction step wherein second extraction unit extracts asecond parameter from the quantitative phase image in the alignment withthe reflection interference image by the image alignment unit.
 15. Thecell observation method according to claim 14, further comprising: acontour extraction step wherein contour extraction unit extracts acontour of the cell, based on the quantitative phase image; a contourapplication step wherein contour application unit applies the contourextracted by the contour extraction unit, to the reflection interferenceimage, to generate a reflection interference image after contourapplication; and a third extraction step wherein third extraction unitextracts a third parameter from the reflection interference image aftercontour application.